[unreadable] Germ cells form upon differentiation of human embryonic stem cells (hESCs) into embryoid bodies (EBs); however, this process is stochastic, rather than a directed differentiation event. The Aims of this proposal are to define conditions that promote germ cell (GC) formation from ESCs exclusively and predictably and to create a stem cell niche in vitro that supports self-renewal and maintenance of human germ cells. As pluripotent hESCs have been derived from GCs in vitro, defining these conditions could indirectly provide a source of hESCs to be studied and manipulated in vitro without the derivation and destruction of human embryos. In addition, these studies will provide a system for elucidating the molecular machinery necessary for establishing the germ cell lineage in humans. Methods: hESC lines (NIH codes: UC01, WA01, and WA13) will be differentiated into EBs, and GC differentiation will be verified by expression of germ cell markers, such as Vasa. To enhance GC differentiation upon embryoid body formation, the ESCs will be cultured on feeder layers that have been demonstrated to support murine germ cell and human primordial germ cell (PGC) proliferation. In addition, growth factors known to support GC and PGC formation, proliferation, and self-renewal, such as BMPs and GDNF, will be added to media after shifting to differentiation conditions. GCs will be purified from the pool of differentiated cells using expression of vasa-GFP transgene. Subsequently, the germ cells will be re-plated into defined growth conditions to encourage germ cell proliferation or differentiation. Environment: The PI previously characterized the germline stem cell niche in the Drosophila testis, establishing a paradigm useful for the identification of stem cell niches in other systems. The proposed research would employ this knowledge to establish conditions for sustained culture of human SSCs. The project will utilize resources available in her laboratory at the Salk Institute and will be supported by interactions with scientists currently studying hESC biology in her department. To familiarize herself with standard methods for the use and propagation of hESCs, the PI plans to attend the UCSF's hESC Training Program. Lay Summary: Tissue replacement therapies will rely on understanding how to make and maintain specific, specialized cell types in culture. This project proposes to develop conditions to continually grow and manipulate one type of stem cell, human germ cells, in the lab. [unreadable] [unreadable]